Biosensor having improved hematocrit and oxygen biases

ABSTRACT

The present invention provides reagent compositions, and analyte measuring devices and methods that utilize the reagent compositions.

CROSS-REFERENCE

This application is a continuation-in-part application of Ser. No.10/278,657, filed Oct. 23, 2002, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

All biosensors for determining the concentration of analytes in a sampleof blood suffer from hematocrit sensitivity to some degree. Thebiosensor response decreases as the hematocrit of the sample increases.There is no single reason for this decrease in the signal, though someof the reasons include diminished diffusion of the analyte in the sampleand increased solution resistance. One of the methods proposed for theelimination of hematocrit sensitivity is to filter the red cells fromthe sample. The membrane technology to filter red cells increases boththe assay time and measurement imprecision. Oxygen sensitivity haspresented a challenge. Biosensors employing the enzyme glucosedehydrogenase are not expected to be oxygen sensitive. However, theoxidation-reduction reactions of the mediator (or coenzyme) couldinvolve free radical intermediates. When these intermediates have longlifetimes, molecular oxygen can quench them, thereby rendering thechemistry sensitive to oxygen tension.

U.S. Pat. Nos. 5,708,247 and 5,951,836 describe a disposable glucosetest strip for use in a test meter of the type that receives adisposable test strip and a sample of blood from a patient and performsan electrochemical analysis. The working formulation comprises a filler,an enzyme effective to oxidize glucose, e.g., glucose oxidase, and amediator effective to transfer electrons from the enzyme. The workingformulation is printed over a conductive base layer to form a workingelectrode. The filler, for example, a silica filler, is selected to havea balance of hydrophobicity and hydrophilicity such that on drying itforms a two-dimensional network on the surface of the conductive baselayer. The response of this test strip is claimed to be temperatureindependent over relevant temperature ranges and is substantiallyinsensitive to the hematocrit of the patient.

In photometric biosensors, a membrane is typically used to separate redcells from a sample of whole blood. The use of a membrane increases thetime of response. U.S. Pat. No. 6,271,045 describes a photometricbiosensor that employs a correction method to compensate for hematocritsensitivity. The biosensor comprises a support member that contains aspreading layer and a reagent layer, and a capillary tube incommunication with the support layer and spreading layer fortransporting a sample of body fluid thereto. A capillary tube isprovided on the support member whereby a fluid containing an analyte tobe tested is introduced into the tube and flows through the tube to thespreading layer and contacts the reagent layer. In order to compensatefor hematocrit level in the case of whole blood, additional sensors canbe implemented so that they inspect the capillary tube in the testdevice, one sensor at the beginning of the capillary channel and one atthe end. In this biosensor, whole blood is applied to the capillarychannel. The entry flow of whole blood is timed as it moves betweensensors. The time that the blood takes to travel the length of thecapillary tube is an indication of the hematocrit of the blood. Thatinformation is used to correct any shift in reflectance readings of theinstrument caused by the hematocrit level. It is also known that theabsorbance of hemoglobin can be measured, and the measurement can beused to account for the sensitivity of the measurement to hemoglobin.

The majority of electrochemical biosensors do not use membranetechnology; hence, electrochemical biosensors suffer from hematocritsensitivity. U.S. Pat. No. 6,284,125 describes a biosensor insensitiveto hematocrit, where red cells are separated from plasma. U.S. Pat. No.6,287,451 describes a biosensor that can employ a method in whichhematocrit level can be measured electrochemically, and the correctedconcentration of an analyte can be determined from the measuredconcentration of the analyte along with factors that depend on thesensitivity of the biosensor to hematocrit level. The magnitude of thehematocrit sensitivity is dependent on the type of biosensor and on thetype of measurement. For example, if the reaction is allowed to go tocompletion, the lengthy reaction time allows for complete oxidation ofthe analyte in the sample, thereby making the measurement less sensitiveto hematocrit.

U.S. Ser. No. 09/529,617, filed Jun. 7, 2000, incorporated herein byreference, describes NAD⁺-dependent and NAD(P)⁺-dependent enzymes havingsubstrates of clinical value, such as glucose, D-3-hydroxybutyrate,lactate, ethanol, and cholesterol. Amperometric electrodes for detectionof these substrates and other analytes can be designed by incorporatingthis class of enzymes and establishing electrical communication with theelectrodes via the mediated oxidation of the reduced cofactors NADH andNADPH. NAD⁺-dependent glucose dehydrogenase can be used as the enzymeand 1,10-phenanthroline-5,6-dione isomer can be used as the mediator.This combination shows hematocrit sensitivity and oxygen sensitivity.The enzyme is not dependent on oxygen (oxygen does not act as aco-substrate as it does with glucose oxidase) and hence is expected tobe insensitive to oxygen. However, the mediator reaction appears to beslow and hence is affected by the presence of oxygen. The mediationreaction involves free radical intermediates. If the reaction is slow,the free radical intermediates have longer half-life; hence, theprobability of being quenched by molecular oxygen is high. Accordingly,the enzyme mediator combination shows oxygen dependency. The hematocritbias of 1,10-phenanthroline-5,6-dione mediator is not clearlyunderstood; however, it is speculated that the slow reaction rate of themediator is responsible for significant hematocrit sensitivity.4,7-Phenanthroline-5,6-dione does not exhibit as much sensitivity tovariations in hematocrit or oxygen as does1,10-phenanthroline-5,6-dione. However, the structure of1,10-phenanthroline-5,6-dione renders it easier to synthesize than doesthe structure of 4,7-phenanthroline-5,6-dione. The starting materialsfor the synthesis of 1,10-phenanthroline-5,6-dione are much lessexpensive than are the starting materials for4,7-phenanthroline-5,6-dione. Additionally, the reaction conditions forthe synthesis of 1,10-phenanthroline-5,6-dione are much less severe thanare the reaction conditions for 4,7-phenanthroline-5,6-dione.Accordingly, it would be desirable to reduce the sensitivity of1,10-phenanthroline-5,6-dione to hematocrit sensitivity and oxygensensitivity.

Glucose monitoring devices are calibrated at normal hematocrit. Insamples having a lower hematocrit, the biosensor reads a higher thanappropriate blood glucose level, and in samples having a higherhematocrit, the biosensor reads a lower than appropriate blood glucoselevel.

SUMMARY OF THE INVENTION

This invention involves a reagent composition that includes a mediator,such as an isomer of phenanthroline quinone (PQ), such as1,10-phenanthroline-5,6-dione or a derivative thereof, a counter anion,and at least one metal ion selected from the group consisting of atransition metal ion, such as, for example, nickel, manganese, iron,osmium, ruthenium, and the like, and heavier alkaline earth metal ion,such as, for example, calcium, barium, and the like. In certainembodiments, the reagent composition further includes an enzymedependent upon NAD(P)⁺, such as, for example, glucose dehydrogenase. Insuch embodiments, when used with a biosensor, the reagent compositionprovides for improved hematocrit bias and oxygen bias. In addition, theelectrodes of biosensors employing this reagent composition provide anaccurate clinical response over a hematocrit range that ranges fromabout 20% to about 70% and over an oxygen tension range that ranges fromabout 1 kPa to about 20 kPa.

Although oxidation of glucose catalyzed by glucose dehydrogenase is notoxygen sensitive, the mediator can be sensitive to oxygen. The1,10-phenanthroline-5,6-dione mediator has the structural formula:

The use of 1,10-phenanthroline-5,6-dione mediator in a glucose biosensoris described in U.S. Ser. No. 09/529,617, filed Jun. 7, 2000, now U.S.Pat. No. 6,736,957, the disclosure of which is incorporated herein byreference in its entirety.

In one aspect, this invention provides a reagent composition for use inan analyte sensing device, wherein the reagent composition includes amediator, wherein the mediator is 1,10-phenanthroline-5,6-dione or aderivative thereof, at least one metal ion, and a counter anion. Themetal ion may be a transition metal ion or a heavier alkaline earthmetal ion.

In another aspect, this invention provides a biosensor e.g., in the formof a strip, wherein the biosensor includes:

an electrode support having at least one electrode thereon; and

a reagent composition adjacent to the at least one electrode, thereagent composition including an enzyme, a 1,10-phenanthroline-5,6-dioneor a derivative thereof as a mediator, a metal ion, wherein the metalion is a transition metal ion or a heavier alkaline earth metal ion, anda counter anion.

In yet another aspect, this invention provides a biosensor, e.g., in theform of a strip, wherein the biosensor has a working electrode thatincludes a reagent composition that includes a NAD(P)⁺-dependent enzyme,1,10-phenanthroline-5,6-dione or a derivative thereof as a mediator, ametal ion, and a counter anion, wherein the metal ion is a transitionmetal ion or a heavier alkaline earth metal ion. In one embodiment, thebiosensor contains an electrode arrangement that includes twoelectrodes. The biosensor includes:

at least one electrode support;

a first electrode disposed on the electrode support, the first electrodeincluding a working area;

a second dual-purpose reference/counter electrode disposed on the sameelectrode support as the first electrode or a different electrodesupport, the dual-purpose reference/counter electrode being spaced apartfrom the first electrode. As such, in certain embodiments, wherein thefirst and second electrodes are on the same electrode support, theelectrodes are in a coplanar configuration. In other embodiments,wherein the first and second electrodes are on different electrodesupports, the electrodes are in a facing configuration. The term “facingelectrodes” refers to a configuration of the working andreference/counter electrodes in which the working surface of the workingelectrode is disposed in approximate opposition to a surface of thereference/counter electrode.

In yet another embodiment, the biosensor contains an electrodearrangement that includes three electrodes, wherein the biosensorincludes:

at least one electrode support;

a first electrode disposed on the electrode support, the first electrodebeing a working electrode;

a second electrode disposed on the same electrode support as the firstelectrode or a different electrode support, the second electrode being areference electrode;

a reagent composition deposited over the first electrode and secondelectrode, wherein the reagent composition comprises an enzyme, amediator, a metal ion, and a counter anion, wherein the mediator is1,10-phenanthroline-5,6-dione or a derivative thereof, and wherein themetal ion is a transition metal ion or a heavier alkaline earth metalion; and optionally, a third electrode disposed on the same electrodesupport as the first electrode or a different electrode support, thethird electrode being a counter electrode, the counter electrodeincludes an electrically conductive material.

Embodiments of the invention described herein provide a mediator that issubstantially insensitive to hematocrit and oxygen, thereby enabling theuse of this mediator in hospital and retail markets, where sampleshaving extreme hematocrit ranges (20% to 70%) and oxygen tensions(neonatal, venous, capillary and arterial) are encountered. A biosensorin the form of a strip employing this mediator can be used for numerousanalytes, such as, for example, glucose, ketone bodies, lactate, andalcohol, as well as others.

Embodiments of the invention described herein exhibit severaladvantages/benefits as compared with other biosensors that are beingused for similar purposes. These advantages/benefits may include:

-   -   1. elimination of the requirement of a membrane or any        cross-linked network;    -   2. the ability to employ a kinetic measurement, and        consequently, the elimination of the requirement to drive the        reaction to completion, thereby eliminating the hematocrit        sensitivity;    -   3. the selection of an appropriate reagent        combination—enzyme/mediator/metal or enzyme/metal complex of the        mediator—is responsible for lower hematocrit sensitivity;    -   4. the catalytic and electrochemical activity of the        mediator/metal combination or metal complex of the mediator is        responsible for oxygen and hematocrit insensitivity;    -   5. improved performance is related to the choice of the        combination of mediator and metal ion; and    -   6. the ability to use a single reagent composition for a working        electrode and reference electrode which dispenses with the need        for a separate reference electrode layer, thereby reducing the        number of manufacturing steps.

Before the present invention is described, it is to be understood thatthis invention is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andexemplary methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupercedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “astrip” includes a plurality of such cells and reference to “thecompound” includes reference to one or more compounds and equivalentsthereof known to those skilled in the art, and so forth.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely”,“only” and the like in connection with the recitation of claim elements,or the use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures:

FIG. 1 is a schematic diagram that illustrates a perspective view of abiosensor strip having a working electrode and a dual-purposereference/counter electrode.

FIG. 2 is a schematic diagram that illustrates a perspective view of abiosensor strip having a working electrode, a reference electrode, and acounter electrode.

FIG. 3 is a graph showing electrochemical properties of1,10-phenanthroline-5,6-dione in the absence of manganese chloride(Curve 1) and in the presence of manganese chloride (Curve 2).

FIG. 4 is a graph showing the response of a biosensor as a function ofthe concentration of glucose for three formulations involving themediator.

FIG. 5 is a graph showing the relative signals of biosensors for ofglucose (15 mM sample) as a function of hematocrit for threeformulations involving the mediator. The data are normalized to thesignal at 40% hematocrit.

FIG. 6 is a graph showing the relative oxygen sensitivities ofbiosensors for three formulations involving the mediator. The data arenormalized to 7 kPa.

FIG. 7 is a comparison of the reagent film homogeneity of ultra-solublemediator [Ni(PQ)₃]Cl₂ of the present invention with the PQ mediator.Panel A is an image of a reagent film derived from a solution containing3% [Ni(PQ)₃]Cl₂. Panel B is an image of a reagent composition derivedfrom a suspension containing 3% PQ.

FIG. 8 is a graph showing the cyclic voltammetry properties of PQ (solidline) and [Ni(PQ)₃]Cl₂ (broken line) on blank screen-printed carbonelectrodes versus Ag/AgCl reference. The graph shows that the[Ni(PQ)₃]Cl₂ mediator of the present invention has a similar oxidationpotential (Eox) to the PQ mediator.

FIG. 9 is a graph showing the electrode calibrations at 1, 2, and 3second assay times with [Ni(PQ)₃]Cl₂ and D-3-hydroxybutyratedehydrogenase (HBDH).

FIG. 10 is a graph showing the electrode calibrations at 1, 2, and 3second assay times with [Ni(PQ)₃]Cl₂ and glucose dehydrogenase withnicotinamide adenine dinucleotide (GDH/NAD).

FIG. 11 is a graph showing the electrode calibrations at 1, 2, and 3second assay times with [Ni(PQ)₃]Cl₂ and glucose dehydrogenase withpyrroloquinolinequinone (GDH/PQQ).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

In general, embodiments of the present invention provide reagentcompositions that include a mediator, such as an isomer ofphenanthroline quinone, 1,10-phenanthroline-5,6-dione or a derivativethereof, a counter anion, and a metal ion. The metal ion can be atransition metal ion or a heavier alkaline earth metal ion. In addition,the present invention also provides biosensors that utilize the reagentcomposition, as well as methods of using the biosensors for detecting ananalyte in a sample.

The structural formula of the mediator 1,10-phenanthroline-5,6-dione isshown below:

When the mediator is reduced by the enzyme, dimers or oligomers or bothare formed on account of intermolecular hydrogen bonding between reduced1,10-phenanthroline-5,6-dione molecules. These oligomers are not solublein the reaction medium and hence are not readily regenerated forcontinued mediation. Intermolecular hydrogen bonding of a dimer ofreduced 1,10-phenanthroline-5,6-dione is shown below.

The dimerization or oligomerization can be minimized in several ways.The nitrogen atoms can be blocked by chemical modification. Asubstituent, e.g., an alkyl group, can be added to one or both of thenitrogen atoms in order to prevent the formation of hydrogen bonds.Preventing the formation of hydrogen bonds also increases the solubilityof the mediator in both the oxidized and reduced form. The methylderivative of 1,10-phenanthroline-5,6-dione shows increased solubility.The compound mediates the oxidation of NADH in the biosensor strip, asdescribed in U.S. Ser. No. 09/529,617, filed Jun. 7, 2000, now U.S. Pat.No. 6,736,957, the disclosure of which is incorporated herein byreference in its entirety. The following structural formula illustratesmono-alkylated 1,10-phenanthroline-5,6-dione, where R represents analkyl group, such as, for example, —CH₃ and X represents an anion suchas BF₄ ⁻:

Synthesis of alkylated compounds requires several steps. The alkyl groupis introduced after the 1,10-phenanthroline-5,6-dione is formed. Theoxidation-reduction properties of alkylated1,10-phenanthroline-5,6-dione may not be dependent on metal ionconcentration, which would indicate that the alkylation process hasinhibited the formation of intermolecular hydrogen bonds.

The nitrogen atoms can also be blocked by the formation of a complexhaving a coordination bond between a ligand and a metal ion. Complexescan be formed prior to being used in a formulation in the strip;alternatively, metal ions can simply be mixed with the ink formulationthat contains the mediator.

As used herein, the expression “transition metal” means those elementsof a metallic nature that have partially filled d or f shells in any oftheir commonly occurring oxidation states. The expression “heavieralkaline earth metals” means those elements of a metallic nature thatare in the IIA column of the periodic table and that have an atomicnumber equal to or higher than 20.

The metal ions suitable for use in this invention include, but are notlimited to, nickel, manganese, zinc, calcium, iron, ruthenium, cobalt,osmium, nickel, copper, rhenium, rhodium, iridium, chromium, technetium,barium, strontium. The binding efficiencies in these complexes aredependent on the particular metal ion employed. For example, Mn (II)ions provide stronger binding than do Mg (II) ions.

A representative metal complex of 1,10-phenanthroline-5,6-dione is:

wherein M is selected from the group consisting of nickel, manganese,iron, cobalt, osmium, ruthenium, calcium, strontium, and barium.

In certain embodiments, the metal is nickel and the nickel complex of1,10-phenanthroline-5,6-dione is:

The generic formula of the complex cation is shown below. The ligands a,b, c, and d can represent two 1,10-phenanthroline-5,6-dione molecules orother monodentate ligands, such as, for example, chloride, water,ammonia, or the like, or multidentate ligands, such as, for example,bipyridyl or the like, and M is selected from the group consisting ofnickel, manganese, iron, cobalt, osmium, ruthenium, calcium, strontium,and barium.

Counter anions suitable for use in this invention include, but are notlimited to, a halide, such as chloride, bromide, fluoride, or iodide, anitrate, a nitrite, a sulfate, a carbonate, a phosphate, a thiocyanate,an acetate, a formate, a citrate, a succinate, an oxalate, a tartrate, abenzoate, an alkyl or aromatic sulfonate, a tungstate, a molybdate, aferricyanide, a nitroprusside, a tetraphenylborate, an anionic dye andan anionic surfactant.

Biosensor strips suitable for this invention are illustrated in FIGS. 1and 2. Referring to FIG. 1, a biosensor strip 10 comprises an electrodesupport 12, e.g., an elongated strip of polymeric material (e.g.,polyvinyl chloride, polycarbonate, polyester, or the like) supportsthree tracks 14 a, 14 b, and 14 c of electrically conductive ink, e.g.,including carbon. These tracks 14 a, 14 b, and 14 c determine thepositions of electrical contacts 16 a, 16 b, and 16 c, a dual-purposereference/counter electrode 18, a working electrode 20, and a triggerelectrode 22. The electrical contacts 16 a, 16 b, and 16 c can beinserted into an appropriate measurement device (not shown) formeasurement of current.

Each of the elongated portions of the conductive tracks 14 a, 14 b, and14 c can optionally be overlaid with a track 24 a, 24 b, and 24 c ofconductive material, e.g., made of a mixture including silver particlesand silver chloride particles. The enlarged exposed area 25 of track 24b overlies the dual-purpose reference/counter electrode 18. A layer of ahydrophobic electrically insulating material 26 further overlies thetracks 14 a, 14 b, and 14 c. The positions of the dual-purposereference/counter electrode 18, the working electrode 20, the triggerelectrode 22, and the electrical contacts 16 a, 16 b, and 16 c are notcovered by the layer of hydrophobic electrically insulating material 26.This hydrophobic electrically insulating material 26 serves to preventshort circuits. Because this insulating material is hydrophobic, it cancause the sample to be restricted to the exposed electrodes. Anexemplary insulating material is commercially available “POLYPLAST”(Sericol Ltd., Broadstairs, Kent, UK).

Optionally, a first layer of mesh 28, a second insulating layer 30, asecond layer of mesh 32, a third insulating layer 34, and a tape 36 canoverlay the hydrophobic insulating material. The tape 36 includes asmall aperture 38 to allow access of the applied sample to theunderlying layers of mesh 28 and 32. The second insulating layer 30 andthe third insulating layer 34 include openings to allow access of theapplied sample to the underlying layers of mesh 28 and 32.

The working electrode 20 includes a layer of conductive materialcontaining a working area 20 a. The working area 20 a may be formed froma reagent composition, which is added (e.g., printed) on the layer ofconductive material of the working electrode 20. The reagent compositionincludes a mixture of an oxidation-reduction mediator, a metal ion, acounter anion, an enzyme, and, optionally, a conductive material.

The working area 20 a may be formed from a printing ink that includesthe reagent composition described above, that includes a mixture of anenzyme, an oxidation-reduction mediator, a counter anion, a metal ion,and, optionally, a conductive material. Alternatively, instead of anenzyme, the working area 20 a can contain a substrate that iscatalytically reactive with an enzyme to be assayed. The reagentcomposition is then applied to the working electrode 20 and thedual-purpose reference/counter electrode 18 as discrete areas of fixedlength. In certain embodiments, the conductive material includesparticles of carbon and the oxidation-reduction mediator comprises1,10-phenanthroline-5,6-dione.

In other embodiments, the electrodes are formed on one or more electrodesupports by any suitable method including chemical etching, laserablation, photolithography, and the like. In general, the electrodesupport is formed from an insulating material, so that it will notprovide an electrical connection between the electrodes of the electrodeset. Examples include glass, ceramics and polymers. In certainembodiments, the electrode substrate is a flexible polymer, such as apolyester or polyimide.

For example, in the laser ablation process, the metallic layer may beablated into an electrode pattern. Furthermore the patterned metalliclayer may be coated or plated with additional metal layers. For example,the metallic layer may be copper, which is then ablated with a laser,into an electrode pattern; subsequently, the copper may be plated with atitanium/tungsten layer, and then a gold layer, to form the desiredelectrodes. In certain embodiments, however, only a single layer of goldis used, which is directly in contact with the electrode substrate. Insuch embodiments, the reagent composition can be positioned adjacent tothe electrode(s).

In one such method, one or more channels are formed in the substrate,for example by an embossing process using an embossing die or roller.Other methods for forming the channels, such as the use of a laser, orphotolithography and etching of the substrate can also be employed ifdesired.

The conductive material may contain pure metals or alloys, or othermaterials which are metallic conductors. Examples include aluminum,carbon (such as graphite), cobalt, copper, gallium, gold, indium,iridium, iron, lead, magnesium, mercury (as an amalgam), nickel,niobium, osmium, palladium, platinum, rhenium, rhodium, selenium,silicon (such as highly doped polycrystalline silicon), silver,tantalum, tin, titanium, tungsten, uranium, vanadium, zinc, zirconium,mixtures thereof, and alloys or metallic compounds of these elements. Incertain embodiments, the conductive material includes carbon, gold,platinum, palladium, iridium, or alloys of these metals, since suchnoble metals and their alloys are unreactive in biological systems.

The reagent composition includes an aqueous suspension of the conductivematerial, a redox mediator, a metal ion, and a counter anion. For theworking electrode 20, the reagent composition also includes an enzyme.For example, when the analyte to be measured is glucose in blood, theenzyme is glucose dehydrogenase, and the redox mediator is a1,10-phenanthroline-5,6-dione. In the alternative, for the workingelectrode 20, the printing ink can include a substrate in lieu of anenzyme when the analyte to be measured is an enzyme.

In certain embodiments, the reagent composition can be screen-printed.In such embodiments, the reagent composition can further include apolysaccharide (e.g., a guar gum or an alginate), a hydrolyzed gelatin,an enzyme stabilizer (e.g., glutamate or trehalose), a film-formingpolymer (e.g., a polyvinyl alcohol), a conductive filler (e.g., carbon),a defoaming agent, a buffer, or a combination of the foregoing.

The electrodes cannot be spaced so far apart that both the workingelectrode 20 and the dual-purpose reference/counter electrode 18 cannotbe covered by the sample. In certain embodiments, the length of the pathto be traversed by the sample (i.e., the sample path) is kept as shortas possible in order to minimize the volume of sample required. Themaximum length of the sample path can be as great as the length of thebiosensor strip. However, the corresponding increase in resistance ofthe sample limits the length of the sample path to a distance thatallows the necessary response current to be generated. The resistance ofthe sample is also influenced by the distance from the edge of the areaof the dual-purpose reference/counter electrode 18 to the edge of theworking area of the working electrode 20. Reducing this distance bypositioning the dual-purpose reference/counter electrode 18 downstreamfrom the working electrode 20 increases the resistance of the sample.Positioning the electrodes contiguously is conventional.

The trigger electrode 22 can be placed downstream of the referenceelectrode. The trigger electrode 22 can be used to determine when thesample has been applied to the strip, thereby activating the assayprotocol. See U.S. Ser. No. 09/529,617, filed Jun. 7, 2000, now U.S.Pat. No. 6,736,957, the disclosure of which is incorporated herein byreference in its entirety.

A biosensor strip 110 suitable for this invention is illustrated in FIG.2. Referring to FIG. 2, an electrode support 111, such as an elongatedstrip of polymeric material (e.g., polyvinyl chloride, polycarbonate,polyester, or the like) supports three tracks 112 a, 112 b, and 112 c ofelectrically conductive ink, such as carbon. These tracks 112 a, 112 b,and 112 c determine the positions of electrical contacts 114 a, 114 b,and 114 c, a reference electrode 116, a working electrode 118, and acounter electrode 120. The electrical contacts 114 a, 114 b, and 114 care insertable into an appropriate measurement device (not shown) formeasurement of current.

Each of the elongated portions of the conductive tracks 112 a, 112 b,and 112 c can optionally be overlaid with a track 122 a, 122 b, and 122c of conductive material, for example made of a mixture including silverparticles and silver chloride particles. The enlarged exposed area oftrack 122 b overlies the reference electrode 116. A layer of ahydrophobic electrically insulating material 124 further overlies thetracks 112 a, 112 b, and 112 c. The positions of the reference electrode116, the working electrode 118, the counter electrode 120, and theelectrical contacts 114 a, 114 b, and 114 c are not covered by the layerof hydrophobic electrically insulating material 124. This hydrophobicelectrically insulating material 124 serves to prevent short circuits.The layer of hydrophobic electrically insulating material 124 has anopening 126 formed therein. This opening 126 provides the boundary forthe reaction zone of the biosensor strip 110. Because this insulatingmaterial is hydrophobic, it can cause the sample to be restricted to theportions of the electrodes in the reaction zone. The working electrode118 comprises a layer of a non-reactive electrically conductive materialon which is deposited a layer 128 containing a reagent composition forcarrying out an oxidation-reduction reaction. At least one layer of mesh130 overlies the electrodes. This layer of mesh 130 protects the printedcomponents from physical damage. The layer of mesh 130 also helps thesample to wet the electrodes by reducing the surface tension of thesample, thereby allowing it to spread evenly over the electrodes. Acover 132 encloses the surfaces of the electrodes that are not incontact with the electrode support 111. This cover 132 is a liquidimpermeable membrane. The cover 132 includes a small aperture 134 toallow access of the applied sample to the underlying layer of mesh 130.

The reagent composition 128 is deposited on that portion of theelectrically conductive material of the working electrode 118 where theoxidation-reduction reaction is to take place when a sample isintroduced to the biosensor strip 110. In such embodiments, the reagentcomposition 128 can be applied to the working electrode 118 as adiscrete area having a fixed length. Typical analytes of interestinclude, for example, glucose and ketone bodies. Typical non-reactiveelectrically conductive materials include, for example, carbon,platinum, palladium, iridium, and gold. A semiconducting material suchas indium doped tin oxide can be used as the non-reactive electricallyconductive material. In certain embodiments, the reagent compositionincludes a mixture of an oxidation-reduction mediator and an enzyme.Alternatively, instead of an enzyme, the reagent composition can containa substrate that is catalytically reactive with an enzyme to be assayed.In the biosensor strips of this invention, the reagent(s) are applied inthe form of a composition containing particulate material and havingbinder(s), and, accordingly, does not dissolve rapidly when subjected tothe sample. In view of this feature, the oxidation-reduction reactionwill occur at the interface of working electrode 118 and the sample. Theglucose molecules diffuse to the surface of the working electrode 118and react with the enzyme/mediator mixture.

In addition to being applied to the working electrode 118, a layer ofthe reagent composition can be applied to any of the other electrodes,such as the reference electrode when desired, as a discrete area havinga fixed length.

Other possible biosensor strip designs include those in which the meshlayer 130 is eliminated, and the flow channel is of such dimensions thatthe biosensor strip takes up a liquid sample by capillary attraction.See U.S. Ser. No. 10/062,313, filed Feb. 1, 2002, incorporated herein byreference.

The mediator can be used for any NAD(P)⁺ dependent enzyme.Representative examples of these enzymes are set forth in Table 1. TABLE1 E.C. (enzyme classification) Number Enzyme name 1.1.1.1 Alcoholdehydrogenase 1.1.1.27 Lactate dehydrogenase 1.1.1.31 β-hydroxybutyratedehydrogenase 1.1.1.49 Glucose-6-phosphate dehydrogenase 1.1.1.47Glucose dehydrogenase 1.2.1.46 Formaldehyde dehydrogenase 1.1.1.37Malate dehydrogenase 1.1.1.209 3-hydroxysteroid dehydrogenase

Other enzyme systems that can be used with the mediator include, but arenot limited to, oxidases (glucose oxidase, cholesterol oxidase, lactateoxidase). Formulations for screen-printing reagents on an electrodecomprise the components set forth in Table 2 and Table 3, where % means% by weight. TABLE 2 (NAD)P⁺ dependent enzyme (such as 200 to 4000 unitsper gram glucose dehydrogenase) Nicotinamide adenine dinucleotide (NAD)5 to 30% 1,10-phenanthroline-5,6-dione 0.1 to 1.5% Filler (such ascarbon or silica) 10 to 30% Binder (such as hydroxyethyl cellulose or0.01 to 0.5% guar gum or alginate) Protein stabilizer (such as trehaloseor 0.01 to 2% bovine serum albumin) Metal ion 0.1 to 10% Buffers andother electrolytes 1 to 10%

TABLE 3 (NAD)P⁺ dependent enzyme (such as 200 to 4000 units per gramglucose dehydrogenase) Nicotinamide adenine dinucleotide (NAD) 5 to 30%Metal complex of 0.1 to 1.5% 1,10-phenanthroline-5,6-dione Filler (suchas carbon or silica) 10 to 30% Binder (such as hydroxyethyl cellulose or0.01 to 0.5% guar gum or alginate) Protein stabilizer (such as trehaloseor 0.01 to 15% bovine serum albumin) Buffers and other electrolytes 1 to10%

The performance of biosensors for determining electrochemical ketonebodies can also be enhanced with the use of this chemistry. A typicalformulation for determination of ketone bodies is shown in Table 4.TABLE 4 β-hydroxybutyrate dehydrogenase 200 to 4000 units per gramNicotinamide adenine dinucleotide (NAD) 5 to 30%1,10-phenanthroline-5,6-dione 0.1 to 1.5% Filler (such as carbon orsilica) 10 to 30% Binder (such as hydroxyethyl cellulose or 0.01 to 0.5%guar gum or alginate) Protein stabilizer (such as trehalose or 0.01 to2% bovine serum albumin) Metal ion 0.1 to 10% Buffers and otherelectrolytes 1 to 10%

In general, NAD(P)⁺-dependent enzymes react with substrate according tothe relationshipRH₂+NAD(P)⁺→R+NAD(P)H+H⁺NAD(P)H is oxidized back to NAD(P)⁺ by the mediator described in thisinvention. The rate of this oxidation reaction is slower than that ofother isomers (1,7-phenanthroline-5,6-dione and4,7-phenanthroline-5,6-dione). This slow reaction rate prevents rapidregeneration of the coenzyme and hence makes it susceptible to variationin hematocrit or oxygen in the sample. The mediator will have higherprobability of reacting with molecular oxygen and hence become sensitiveto oxygen. The diffusion of the mediator in the sample is affected bythe hematocrit variation and slow reacting mediator will be moreaffected by restricted mobility compared to a fast reacting mediator.The metal ions described herein allow rapid regeneration of the coenzymeand hence makes it less susceptible to variation in hematocrit or oxygenin the sample.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1

Metal ion is required for efficient mediation of NADH oxidation by1,10-phenanthroline-5,6-dione. In solution,1,10-phenanthroline-5,6-dione does not show any electrochemicaloxidation at physiological pH conditions. In the presence of a metal ionsuch as manganese, the mediator shows both oxidation and reductioncurrent. FIG. 3 shows the electrochemical properties of1,10-phenanthroline-5,6-dione in the presence of manganese chloride(Curve 2) and in the absence of manganese chloride (Curve 1).

The concentration of the metal ion required for the optimal performanceof the biosensor depends on the binding constant of the metal and the1,10-phenanthroline-5,6-dione. The efficiency of complex formation andstability of the complex is dependent on the metal ion. For example,only 10 mM manganese chloride is sufficient to achieve the performancethat is achieved by a 360 mM magnesium chloride for 30 mM of1,10-phenanthroline-5,6-dione in the formulation. Ten (10) mM manganesechloride corresponds to a ratio of one (1) metal ion to three (3)1,10-phenanthroline-5,6-dione molecules in the formulation that formsthe metal complex. The binding constant of Pb (II) with1,10-phenanthroline-5,6-dione is greater than the binding constant of Mn(II) or Mg (II) with 1,10-phenanthroline-5,6-dione; however, the enzymeis inactivated by Pb (II). Mediation of NADH oxidation by1,10-phenanthroline-5,6-dione in the presence of other transition metalions and heavier alkaline earth metal ions has been demonstrated.

Transition metal ions and heavier alkaline earth metal ions can also beused as complexes for the mediation of NADH oxidation. The performanceof the free ion Mn (II) mixed in the formulation is identical to theperformance of the complex that is formed before it is added to the inkformulation.

The hematocrit and oxygen bias of formulations containing Mn (II) aresignificantly improved compared to the formulations containing Mg (II).FIG. 4 shows correlation of biosensor response as a function ofconcentration of glucose for the three mediation chemistries. FIG. 5shows the relative signals of a 15 mM sample as a function of hematocritnormalized to the signal at 40% hematocrit. FIG. 6 shows oxygensensitivities of the biosensors with three chemistries normalized to 7kPa. Similar hematocrit and oxygen bias advantages are seen with the Fe(II) complex of 1,10-phenanthroline-5,6-dione. In other words, using atransition metal ion or a heavier alkaline earth metal ion in theformulation improves the electrochemical properties of the compound.Some of the transition metal ions and heavier alkaline earth metal ionsshow improved oxygen and hematocrit sensitivities as compared with othertransition metal ions and heavier alkaline earth metal ions.

The complexes were either formed prior to use in the strip or the metalions were mixed with the ink. The metal ions used were transition metalions and heavier alkaline earth metal ions.

Example 2

Synthesis

Ultra-soluble [Ni(PQ)₃]²⁺ salts were prepared by adding solid PQ (3-foldexcess) to a solution of the appropriate nickel (II) salt. PQ wasdissolves to form the Ni(II) complex. The solution was filtered and thenfreeze-dried to give the solid [Ni(PQ)₃]²⁺ salt.

Slightly soluble [Ni(PQ)₃]²⁺ salts were prepared by adding theappropriate anion to a solution of an ultra-soluble [Ni(PQ)₃]²⁺ salt,e.g., [Ni(PQ)₃]Cl₂ then filtering the resulting precipitated productfollowed by drying.

Aqueous Solubility

Solubility tests in water were performed on the new redox mediators incomparison with the standard PQ mediators. Of the new mediators preparedso far, the fluoride, chloride, bromide, iodide, nitrate, sulfate andacetate salts of [Ni(PQ)₃]²⁺ were found to have aqueous solubility inexcess of 20 mg/ml (2%) compared to <4 mg/ml (<0.4%) for the standard PQmediators. As expected, the perchlorate, tetrafluroborate andhexafluorophosphate salts of [Ni(PQ)₃]²⁺ had very low solubility inwater (<0.5 mg/ml). A summary of the results is provided in Table 5.

Table 5 TABLE 5 Mediator Solubility in Water [Ni(PQ)₃]Cl₂ >400 mg/ml(>40%) [Ni(PQ)₃]Br₂ 20 mg/ml (2%) [Ni(PQ)₃]I₂ 20 mg/ml (2%)[Ni(PQ)₃]F₂ >200 mg/ml (>20%) [Ni(PQ)₃](NO₃)₂ 50 mg/ml (5%)[Ni(PQ)₃](ClO₄)₂ <0.5 mg/ml (<0.05%) [Ni(PQ)₃](PF₆)₂ <0.5 mg/ml (<0.05%)[Ni(PQ)₃](BF₄)₂ <0.5 mg/ml (<0.05%) [Ni(PQ)₃](O₂CCH₃)₂ >500 mg/ml (>50%)[Ni(PQ)₃]SO₄ >500 mg/ml (>50%) PQ 2 mg/ml (0.2%) [Mn(PQ)₃]Cl₂ 4 mg/ml(0.4%) [Fe(PQ)₃]Cl₂ >200 mg/ml (>20%)Reagent Film Homogeneity

A comparison of ultra-soluble mediator [Ni(PQ)₃]Cl₂ of the presentinvention with the standard PQ mediator was also performed. FIG. 7provides results of the comparison of the reagent film homogeneity ofultra-soluble mediator [Ni(PQ)₃]Cl₂ of the present invention with the PQmediator. Panel A is an image of a reagent film derived from a solutioncontaining 3% [Ni(PQ)₃]Cl₂. Panel B is an image of a reagent compositionderived from a suspension containing 3% PQ. As shown in FIG. 7, the[Ni(PQ)₃]Cl₂ mediator provides a uniform reagent composition with novisible sign of solid particles or crystals whereas the reagentcomposition derived from the PQ mediator display a non-even distributionof solid particles.

Electrochemistry

The electrochemistry properties the ultra-soluble mediator [Ni(PQ)₃]Cl₂of the present invention and the standard PQ mediator were alsodetermined. The graph of FIG. 8 shows the cyclic voltammetry propertiesof PQ (solid line) as compared to the properties of [Ni(PQ)₃]Cl₂ (brokenline) on blank screen-printed carbon electrodes versus Ag/AgClreference. The graph shows that the [Ni(PQ)₃]Cl₂ mediator of the presentinvention has a similar oxidation potential (Eox) to the standard PQmediator.

Examples of Electrode Calibrations with Different Enzymes

A stock aqueous reagent composition for use in analyte sensing wasprepared containing the following:

3.4% [Ni(PQ)₃]Cl₂,

2% polymer,

4.7% trehalose,

0.7% MgCl₂, and

0.7% NAD (pH adjusted to 7).

Various enzymes were then added and the resulting solution was used tocoat gold electrodes which were dried for 3 min at 75° C. Pre-formedcapillary fill cells were then applied and the strips trimmed to size.Testing was performed using the appropriate substrate/analyte solutionsin PBS. Test parameters included the following: 0 sec delay, +200 mVapplied potential (vs. mediator redox couple—biamperometry), 100 Hzsampling time, manual assay start. Electrode calibrations were performedat 1, 2, and 3 sec assay times. The working electrode reaction was basedon:[Ni(PQ)₃]²⁺+NADH=[Ni(PQH₂)₃]²⁺+NADThen [Ni(PQH₂)₃]²⁺ is oxidized to [Ni(PQ)₃]²⁺ at the electrode. Thereference electrode reaction was based on the reduction of [Ni(PQ)₃]²⁺to [Ni(PQH₂)₃]²⁺.

FIG. 9 shows the electrode calibrations at 1, 2, and 3 second assaytimes with [Ni(PQ)₃]Cl₂ and D-3-hydroxybutyrate dehydrogenase (HBDH).FIG. 10 shows the electrode calibrations at 1, 2, and 3 second assaytimes with [Ni(PQ)₃]Cl₂ and glucose dehydrogenase with nicotinamideadenine dinucleotide (GDH/NAD). FIG. 11 shows the electrode calibrationsat 1, 2, and 3 second assay times with [Ni(PQ)₃]Cl₂ and glucosedehydrogenase with pyrroloquinolinequinone (GDH/PQQ). Therefore, asprovided in the figures the reagent composition of the present inventionis suitable for use with a variety of different enzymes for detection ofan analyte.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1. A reagent composition, comprising: a mediator, wherein the mediatoris 1,10-phenanthroline-5,6-dione or a derivative thereof; a metal ion,wherein the metal ion is a transition metal ion or a heavier alkalineearth metal ion; and a counter anion.
 2. The reagent composition ofclaim 1, wherein the metal ion is nickel, manganese, iron, cobalt,osmium, or ruthenium.
 3. The reagent composition of claim 1, wherein themetal ion is calcium, strontium, or barium.
 4. The reagent compositionof claim 1, wherein the metal ion is in the form of a salt.
 5. Thereagent composition of claim 1, wherein the metal ion is nickel.
 6. Thereagent composition of claim 1, wherein the metal ion is in the form ofa complex with the mediator.
 7. The reagent composition of claim 1,wherein the mediator is 1,10-phenanthroline-5,6-dione.
 8. The reagentcomposition of claim 1, wherein the counter anion is a halide, anitrate, a nitrite, a sulfate, a carbonate, a phosphate, a thiocyanate,an acetate, a formate, a citrate, a succinate, an oxalate, a tartrate, abenzoate, or an alkyl or aromatic sulfonate.
 9. The reagent compositionof claim 8, wherein the halide is chloride, bromide, fluoride, oriodide.
 10. The reagent composition of claim 1, wherein the counteranion is a tungstate, a molybdate, a ferricyanide, a nitroprusside, atetraphenylborate, an anionic dye or an anionic surfactant.
 11. Thereagent composition of claim 1, wherein the reagent composition furthercomprises an enzyme.
 12. The reagent composition of claim 11, whereinthe enzyme is glucose oxidase or glucose dehydrogenase.
 13. The reagentcomposition of claim 11, wherein the enzyme is NAD(P)⁺-dependentdehydrogenase.
 14. The reagent composition of claim 11, wherein theenzyme is glucose dehydrogenase.
 15. A biosensor comprising: (a) anelectrode support; (b) a first electrode disposed on the electrodesupport, the first electrode being a working electrode, (c) a secondelectrode disposed on the electrode support, the second electrode beinga reference electrode; and (d) a reagent composition deposited over thefirst electrode and second electrode, wherein the reagent compositioncomprises an enzyme, a mediator, a metal ion, and a counter anion,wherein the metal ion is a transition metal ion or a heavier alkalineearth metal ion, and wherein the mediator is1,10-phenanthroline-5,6-dione or a derivative thereof.
 16. The biosensorof claim 15, wherein the metal ion is nickel, manganese, iron, cobalt,osmium, or ruthenium.
 17. The biosensor of claim 15, wherein the metalion is calcium, strontium, or barium.
 18. The biosensor of claim 15,wherein the metal ion is nickel.
 19. The biosensor of claim 15, whereinthe metal ion is in the form of a salt.
 20. The biosensor of claim 15,wherein the metal ion is in the form of a complex with the mediator. 21.The biosensor of claim 15, wherein the enzyme is a glucose oxidase or aglucose dehydrogenase.
 22. The biosensor of claim 15, wherein the enzymeis NAD(P)⁺-dependent dehydrogenase.
 23. The biosensor of claim 15,wherein the enzyme is glucose dehydrogenase.
 24. The biosensor of claim15, wherein the mediator is 1,10-phenanthroline-5,6-dione.
 25. Thebiosensor of claim 15, wherein the counter anion is a halide, a nitrate,a nitrite, a sulfate, a carbonate, a phosphate, a thiocyanate, anacetate, a formate, a citrate, a succinate, an oxalate, a tartrate, abenzoate, or an alkyl or aromatic sulfonate.
 26. The biosensor of claim25, wherein the halide is chloride, bromide, fluoride, or iodide. 27.The biosensor of claim 15, wherein the counter anion is a tungstate, amolybdate, a ferricyanide, a nitroprusside, a tetraphenylborate, ananionic dye or an anionic surfactant.
 28. The biosensor of claim 15,further comprising a covering layer defining an enclosed space over theelectrodes, the covering layer having an aperture for receiving a sampleinto the enclosed space.
 29. The biosensor of claim 28, furthercomprising a least one layer of mesh interposed in the enclosed spacebetween the covering layer and the electrodes.
 30. The biosensor ofclaim 15, wherein the counter electrode is positioned relative to theworking electrode and the reference electrode such that a liquid samplewill contact the working electrode and the reference electrode prior tocontacting the counter electrode.
 31. The biosensor of claim 15, whereinat least one electrode comprises carbon, palladium, gold, platinum, oriridium.
 32. A biosensor comprising: an electrode support having atleast one gold electrode thereon; and a reagent composition adjacent theat least one gold electrode, the reagent composition comprising: anenzyme, a mediator, wherein the mediator is1,10-phenanthroline-5,6-dione or a derivative thereof, a metal ion,wherein the metal ion is a transition metal ion or a heavier alkalineearth metal ion, and a counter anion.
 33. The biosensor of claim 32,wherein the metal ion is nickel, manganese, iron, cobalt, osmium, orruthenium.
 34. The biosensor of claim 32, wherein the metal ion iscalcium, strontium, or barium.
 35. The biosensor of claim 32, whereinthe metal ion is nickel.
 36. The biosensor of claim 32, wherein themetal ion is in the form of a salt.
 37. The biosensor of claim 32,wherein the metal ion is in the form of a complex with the mediator. 38.The biosensor of claim 32, wherein the enzyme is a glucose oxidase or aglucose dehydrogenase.
 39. The biosensor of claim 32, wherein the enzymeis NAD(P)⁺-dependent dehydrogenase.
 40. The biosensor of claim 32,wherein the enzyme is glucose dehydrogenase.
 41. The biosensor of claim32, wherein the mediator is 1,10-phenanthroline-5,6-dione.
 42. Thebiosensor of claim 32, wherein the counter anion is a halide, a nitrate,a nitrite, a sulfate, a carbonate, a phosphate, a thiocyanate, anacetate, a formate, a citrate, a succinate, an oxalate, a tartrate, abenzoate, or an alkyl or aromatic sulfonate.
 43. The biosensor of claim42, wherein the halide is chloride, bromide, fluoride, or iodide. 44.The biosensor of claim 32, wherein the counter anion is a tungstate, amolybdate, a ferricyanide, a nitroprusside, a tetraphenylborate, ananionic dye or an anionic surfactant.
 45. The biosensor of claim 32,further comprising a covering layer defining an enclosed space over theelectrodes, the covering layer having an aperture for receiving a sampleinto the enclosed space.
 46. The biosensor of claim 45, furthercomprising a least one layer of mesh interposed in the enclosed spacebetween the covering layer and the electrodes.
 47. The biosensor ofclaim 32, wherein the counter electrode is positioned relative to theworking electrode and the reference electrode such that a liquid samplewill contact the working electrode and the reference electrode prior tocontacting the counter electrode.
 48. The biosensor of claim 32, whereinat least one electrode comprises carbon, palladium, gold, platinum, oriridium.
 49. A method for determining the concentration of an analyte ina sample of biological fluid, the method comprising the steps of: (a)inserting a biosensor into an analyte monitor, the biosensor comprising:an electrode support having at least one electrode thereon; and areagent composition adjacent the at least one gold electrode, thereagent composition comprising: an enzyme, a mediator, wherein themediator is 1,10-phenanthroline-5,6-dione or a derivative thereof, ametal ion, wherein the metal ion is a transition metal ion or a heavieralkaline earth metal ion, and a counter anion; (b) applying thebiological fluid to the biosensor; (c) applying a voltage at theelectrode; (d) measuring the current at the electrode; and (e)correlating the current measured to the concentration of the analyte.50. The biosensor of claim 49, wherein the metal ion is nickel,manganese, iron, cobalt, osmium, or ruthenium.
 51. The biosensor ofclaim 49, wherein the metal ion is calcium, strontium, or barium. 52.The biosensor of claim 49, wherein the metal ion is nickel.
 53. Thebiosensor of claim 49, wherein the metal ion is in the form of a salt.54. The biosensor of claim 49, wherein the metal ion is in the form of acomplex with the mediator.
 55. The biosensor of claim 49, wherein theenzyme is a glucose oxidase or a glucose dehydrogenase.
 56. Thebiosensor of claim 49, wherein the enzyme is NAD(P)⁺-dependentdehydrogenase.
 57. The biosensor of claim 49, wherein the enzyme isglucose dehydrogenase.
 58. The biosensor of claim 49, wherein themediator is 1,10-phenanthroline-5,6-dione.
 59. The biosensor of claim49, wherein the counter anion is a halide, a nitrate, a nitrite, asulfate, a carbonate, a phosphate, a thiocyanate, an acetate, a formate,a citrate, a succinate, an oxalate, a tartrate, a benzoate, or an alkylor aromatic sulfonate.
 60. The biosensor of claim 59, wherein the halideis chloride, bromide, fluoride, or iodide.
 61. The biosensor of claim49, wherein the counter anion is a tungstate, a molybdate, aferricyanide, a nitroprusside, a tetraphenylborate, an anionic dye or ananionic surfactant.
 62. The biosensor of claim 49, further comprising acovering layer defining an enclosed space over the electrodes, thecovering layer having an aperture for receiving a sample into theenclosed space.
 63. The biosensor of claim 62, further comprising aleast one layer of mesh interposed in the enclosed space between thecovering layer and the electrodes.
 64. The biosensor of claim 49,wherein the counter electrode is positioned relative to the workingelectrode and the reference electrode such that a liquid sample willcontact the working electrode and the reference electrode prior tocontacting the counter electrode.
 65. The biosensor of claim 49, whereinat least one electrode comprises carbon, palladium, gold, platinum, oriridium.